This set provides 3 vectors (pQE-16, pQE-60, and pQE-70) for expression of C-terminally 6xHis-tagged proteins and is recommended for open reading frames with “pause sites”, which can cause premature termination. pQE-60 and pQE-70 allow the original start codon of the coding fragment to replace the ATG in the pQE vector, preserving the authentic N-terminus of the protein. These two constructs are created by introducing an NcoI and a SphI restriction site, respectively, at the ATG codon of the insert by PCR or mutagenesis. pQE-16 allows expression of C-terminally 6xHis-tagged DHFR-fusion proteins. DHFR (dihydrofolate reductase) enhances antigenicity and stability, and is recommended for poorly expressed proteins or short peptides prone to proteolysis. Since DHFR itself displays little immunogenicity in mouse and rat, DHFR-fusion proteins are ideal for epitope screening.

QIAexpress pQE vectors combine a powerful phage T5 promoter (recognized by E. coli RNA polymerase) with a doublelacoperator repression module to provide tightly regulated, high-level expression of recombinant proteins in E. coli (see figure "QIAexpress pQE vector"). Protein synthesis is effectively blocked in the presence of high levels of lac repressor and the stability of cytotoxic constructs is enhanced. The pQE vectors enable placement of the 6xHis tag at either the N- or C-terminus of the recombinant protein.

Elements present in QIAexpress pQE vectors

Element

Description

Optimized promoter/operator element

Consists of the phage T5 promoter and two lac operator sequences, which increase the probability of lac repressor binding and ensure efficient repression of the powerful T5 promoter

Synthetic ribosomal binding site RBSII

For efficient translation

6xHis-tag coding sequence

Either 5' or 3' to the polylinker cloning region

Translational stop codons

In all reading frames for convenient preparation of expression constructs

Two strong transcriptional terminators

t0 from phage lambda, and T1 from rrnB operon of E. coli, to prevent read-through transcription and ensure stability of the expression construct

ColE1 origin of replication

From pBR322

Beta-lactamase gene (bla)

Confers ampicillin resistance

Procedure

Inserts encoding proteins of interest are cloned into appropriate constructs (for detailed information, see The QIAexpressionist Handbook) and transformed into a suitable E. colistrain for expression. Expression is induced by addition of IPTG. Vector pQE-TriSystem constructs can be transformed intoE. coli, used as a shuttle vector for recombinant protein expression in insect cells, or transfected into mammalian cells.